3.2.1 Unzipping experiments

DNA unzipping can be performed in two different ways: 1) at controlled position or 2) at controlled force. The first one consists in separating both strands and applying whatever force is required to disrupt the bases. It is a sequential process and the disruption (i.e., opening) of bases occurs progressively. The second one consists in increasing the force applied to the strands of the molecule. In this protocol, all the base pairs of the DNA are suddenly disrupted when a critical force is reached. Depending on the experimental setup, the process of unzipping will be of one kind or the other. All these experiments can be performed at different speeds, which will determine whether the process is out of equilibrium or quasistatic.

One of the most important features of optical tweezers is that it allows to control and measure the extension of the DNA and the force applied to its ends. The possibility of measuring the force during the unzipping gives a lot of information about the process. The typical forces exerted by an optical trap are of the order of magnitude of the critical force required to unzip the DNA molecule. Moreover, feedback algorithms are able to control the force at 4 kHz frequency. So both types of unzipping experiments (controlled position or controlled force) can be performed with optical tweezers.

In order to unzip DNA, the synthesized molecular constructs are attached to two homogeneous beads made of polystyrene that have an index of refraction of $ n=1.51$. One bead is a Streptavidin (SA) coated microsphere (2.0-2.9 $ \mu $m; G. Kisker GbR, Products for Biotechnology). The other bead is a protein G microsphere (3.0-3.4 $ \mu $m; Spherotech, Libertyville, IL) coated with anti-digoxigenin (AD) polyclonal antibodies (Roche Applied Science). The SA bead bonds to the biotin-labeled handle of the molecular construct, while the AD bead bonds to the other handle, which is labeled with digoxigenins. The rest of the elements of the molecular construct (ssDNA, dsDNA, loop) do not stick to polystyrene beads. This assures that unzipping experiments are not affected by unintended interactions. The SA bead is usually fixed at the tip of the micropipette by air suction, while the AD bead is captured in the optical trap (see Fig. 3.7).

Figure 3.7: Experimental setup to unzip the DNA molecular constructs. Picture not to scale.

JM Huguet 2014-02-12